Biophysical studies of the interaction between high molecular weight kininogen and gC1q-R : initiating the contact pathway

Slater, Alex (2017) Biophysical studies of the interaction between high molecular weight kininogen and gC1q-R : initiating the contact pathway. PhD thesis, University of Nottingham.

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Abstract

The contact pathway is part of the coagulation pathway involved in haemosta- sis. It is responsible for the deposition of fibrin fibres during blood coagulation as well as the production of the inflammatory mediator bradykinin. This path- way is initiated when the three enzymes prekallikrein, factor XI and factor XII are assembled at the cell surface triggering a cleavage cascade that results in the deposition of fibrin. High molecular weight kininogen is a cofactor respon- sible for the presentation of Factor XI and prekallikrein to the cell surface where these proteins come into contact with the cell bound FXII. Both FXII and high molecular weight kininogen bind to endothelial cells through the monotrimeric endothelial cell receptor gC1q-R in a zinc-dependent manner.

Domain 5 is the cell surface binding domain of high molecular weight kinino- gen. Studies were performed on isolated domain 5 which showed it to be a zinc binding, intrinsically disordered domain that binds directly to trimeric gC1q-R. Isothermal titration calorimetry revealed that gC1q-R binding occurs through a sequential binding mechanism where three domain 5 ligands bind to one trimer. The production of N and C-terminal truncations of domain 5 revealed a zinc-dependent N-terminal binding region and a zinc-independent C-terminal binding region that simultaneously bind separate sites on gC1q-R. The C-terminal binding region was further narrowed down to a Lys rich por- tion of domain 5. Further mutation studies on gC1q-R revealed that the β6-β7 loop, located towards the centre of the cavity, is crucial for D5 binding.

The findings highlighted within this thesis provide structural and mechanistic detail into the complex interaction between domain 5 and gC1q-R. The dual binding of high molecular weight kininogen and factor XII with this receptor is discussed in order to further understand how gC1q-R assembles the contact initiator proteins at the cell surface.

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Searle, Mark
Emsley, Jonas
Subjects: Q Science > QP Physiology > QP501 Animal biochemistry
Faculties/Schools: UK Campuses > Faculty of Science > School of Chemistry
Item ID: 45738
Depositing User: Slater, Alex
Date Deposited: 14 Dec 2017 04:40
Last Modified: 15 Dec 2017 17:20
URI: http://eprints.nottingham.ac.uk/id/eprint/45738

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